Mechanism of antibody-specific deglycosylation and immune evasion by Streptococcal IgG-specific endoglycosidases

Bacterial pathogens have evolved intricate mechanisms to evade the human immune system, including the production of immunomodulatory enzymes. Streptococcus pyogenes serotypes secrete two multi-modular endo-β-N-acetylglucosaminidases, EndoS and EndoS2, that specifically deglycosylate the conserved N-glycan at Asn297 on IgG Fc, disabling antibody-mediated effector functions. Amongst thousands of known carbohydrate-active enzymes, EndoS and EndoS2 represent just a handful of enzymes that are specific to the protein portion of the glycoprotein substrate, not just the glycan component. Here, we present the cryoEM structure of EndoS in complex with the IgG1 Fc fragment. In combination with small-angle X-ray scattering, alanine scanning mutagenesis, hydrolytic activity measurements, enzyme kinetics, nuclear magnetic resonance and molecular dynamics analyses, we establish the mechanisms of recognition and specific deglycosylation of IgG antibodies by EndoS and EndoS2. Our results provide a rational basis from which to engineer novel enzymes with antibody and glycan selectivity for clinical and biotechnological applications.


Supplementary Table 4. Precursors and quantities used for the preparation of [U-15 N, 2 H] MIL proS V proS [ 13 C, 1 H 3 ]-methyl labeled endoglycosidases.
A systematic review on precursors required for optimal methyl group isotope labelling in E. coli can be found somewhere else 1 .

Supplementary
Therefore, we turned to specific 13 C side chain methyl group labeling strategies 1,9,10 , which enabled the acquisition of 1 H, 13 Fig. 16c,d). In addition, the selected methyl probes are uniformly distributed around the putative glycan binding pockets in both enzymes, granting the extraction of CSPs upon ligand titration ( Fig.   6a; Supplementary Fig. 17a). 1 H, 15  Detailed examples from EndoS2 E186L and EndoBT D312A/E314L can be scrutinized in Fig. 6B and in Supplementary Figs. 17c, respectively. Following this methodology, we could safely identify 10 methyl signals reporting for the binding of EndoS2 E186L to CT, CT n-1 and Rituximab-Fc, and another 10 signals reporting for the interaction of EndoBT D312A/E314L with HM (signals 1-10 in Supplementary Fig. 23 and signals 11-20 in Supplementary Fig. 24, respectively). EndoS2 also possesses a calcium-binding pocket located in the b-sandwich (BS) domain (Fig. 6a). Signals exhibiting CSPs greater than 2 standard deviations upon titration with calcium chloride were selected as reporters for calcium binding.

Extraction of kinetic and thermodynamic parameters from methyl-TROSY spectra
Titration of catalytically dead ENGases with carbohydrates CT, CT n-1 or Rituximab-Fc produced CSPs in the slow-exchange regime in the NMR time-scale, leading to varying peak intensities during titration ( Supplementary Figs. 25-30). On the other hand, titration of EndoS2 with CaCl 2 produced CSPs in the fast-exchange regime, which translates into shifts in peak positions ( Supplementary Fig. 31). Methyl-TROSY based titrations in slow exchange were subjected to lineshape analysis with TITAN software package. For each titration point, the corresponding methyl-TROSY spectrum was imported into MATLAB and then analyzed with the TITAN algorithm, applying a two-state ligand binding model. Quantitative lineshape analysis yielded dissociation constants K D and on-and off-rate constants k on and k off . A representative example of lineshape fitting can be found in Fig. 6D.
In the main text, we have described the kinetics of binding of EndoS2 to its substrate CT, Rituximab-Fc and the reaction product CT n-1 . Here, we focus on the effect of calcium in the interaction of EndoS2 with CT and Rituximab-Fc. Calcium binding produced similar CSPs in EndoS2 and EndoS2 E186L species with virtually identical dissociation constants K D , suggesting no influence of the mutation E186L on calcium recognition (Table 1, entries 6,7, Supplementary Fig. 32). It is worth noting however, that some signals showed significant CSPs during the titration of EndoS2 E186L with CT, CT n-1 , Rituximab-Fc and CaCl 2 . Such promiscuity in CSPs suggests the presence of alternative calcium-binding sites or a cross-talking mechanism between the GH and BS domains, as it has already been proposed from functional studies with chimeric ENGases. 2 Here, we did not attempt any further analysis on such signals.
Instead, they will be the subject of a future study, whenever a spectrum assignment is available for EndoS2.  (Supplementary Fig. 18). Cleaved carbohydrate was quantified from the integral of the composite signal at 2.08-2.10 ppm, which reports for N-acetylglucosamine and Nacetylneuraminic acid moieties (Supplementary Fig. 19a). Reaction time-courses were measured to close-to-reaction completion for five Rituximab-Fc initial concentrations ( Supplementary Fig. 19b). The reaction rate was calculated from the fitting of initial velocity rates (# $ ) to the equation of Michaelis-Menten (5) (Fig. 6e). Under these conditions, a catalytic rate k cat of 5.9 s -1 and a Michaelis-Menten constant K M of 26.7 µM were observed. It is important to remark that K M resembles K D only when k off >> k cat , according to Eq. 6. In this study this condition is not met (k off = 4.0 s -1 ≈ k cat = 5.9 s -1 ), indicating that the system is not in rapid equilibrium. Nevertheless, care has to be taken when combining results obtained from different EndoS2 species (wt and E186L), because non-identical protein-carbohydrate interactions can result in slightly different kinetic parameters. In conclusion, this study shows for the first time an overview of enzyme kinetics followed by EndoS2. We hope this information will be key for the future development of ENGases with improved activity and selectivity. trajectories. For aromatic residues and Pro, the center of the aromatic ring and the center of the ring are considered, respectively, for these calculations. For L314, S298 and E354 residues, the Cd, the OH group, and the oxygens of the carboxylate are considered, respectively, for the calculations. Cut-off distance: 5.5 Å -p-p stating and CH-p -, and 3.5 Å -hydrogen bond-. Errors are given as SD. The numbers in red and in parentheses are the experimental values found in the cryo-EM structure. The dashed lines represent the distance values found in the cryo-EM structure of EndoS-Fc.  Global lineshape analysis of a series of concentration dependent methyl-TROSY spectra using TITAN 13 yielded a fitted set of 10 signals. It should be noted that in principle this analysis does not depend on any assignments. Experimental and fitted cross peak are shown in grey and magenta, respectively, reflecting the good quality of the fit. TITAN analysis results in a dissociation constant K D of 106.9 ± 6.7 µM and a dissociation rate constant k off of 11.4 ± 1.5 s -1 .